The proteasome is the enzyme that is primarily responsible for turnover of proteins within the cytoplasm and nucleus of eukaryotic cells. A substrate destined for the proteasome is first conjugated to an ubiquitin chain, which binds to receptors on the proteasome, thereby tethering the substrate so that it can be translocated into the inner chamber of the proteasome where it is degraded. Many clinical studies have confirmed that small molecules that block the protein- degrading active sites in the inner chamber of the proteasome can stem the progression of human cancers including multiple myeloma and mantle cell lymphoma. In prior work, we discovered that the Rpn11 subunit of the proteasome contains a novel zinc metalloprotease active site that removes the ubiquitin chain from proteasome substrates. This 'deubiquitination' catalyzed by Rpn11 is essential for the protein-degrading activity of the proteasome, presumably because the bulky ubiquitin chain prevents substrate from being inserted into the inner chamber of the proteasome. Rpn11 metalloprotease thus represents a new proteasome activity that can potentially be targeted by small molecules to block degradation of proteasome substrates and stem the growth of human tumors. The goal of this application is to use this assay to screen a large compound library (~330,000 compounds) from the MLPCN located at the Conrad Prebys Center for Chemical Genomics to identify Rpn11 inhibitors and triage these inhibitors in secondary and tertiary assays devised to prioritize potent, selective, competitive, reversible and cell permeable hits. Our strategy is based on a substrate comprising four tandem ubiquitins fused to a peptide bearing a unique cysteine that can be labeled with a fluorophore. Cleavage of this substrate by Rpn11 at the junction between the fourth ubiquitin and the peptide releases the low molecular weight fluorescent peptide, resulting in a large depolarization of the fluorophore. By measuring the fluorescence polarization of the substrate in the presence of 26S proteasome plus various compounds, we should be able to identify those compounds, termed hits, that block Rpn11-dependent release of the fluorescent peptide product. Hits that subsequently pass the battery of secondary and tertiary assays will inform follow-up SAR studies to improve compound potency, selectivity and cell-permeability. These inhibitors will be employed to differentiate ubiquitin-dependent and ubiquitin-independent substrates of the proteasome and to test whether inhibition of Rpn11 is a viable strategy for chemotherapy of cancer. PUBLIC HEALTH RELEVANCE: The proteasome is the target for new drugs - 'proteasome inhibitors' - that are used to treat the hematological malignancies multiple myeloma and mantle cell lymphoma. Several years ago, we discovered a new activity of the proteasome that we named Rpn11 isopeptidase. Targeting this activity with drugs could represent a way to block proteasome function in cancer cells that differs from the mechanism of action of the existing proteasome inhibitors. We propose to explore this possibility by a high throughput screening strategy and follow-up medicinal chemistry efforts that will allow us to isolate drugs that block Rpn11 isopeptidase. Drugs that emerge from this effort will then be tested to see if they possess anti-tumor activity using cancer cells grown in culture.